A restricted receptor (R) for interleukin 13 (IL13), IL13Ralpha2, is over-expressed in high-grade (grade III and IV) astrocytoma (HGA) patients. IL13Ralpha2 exhibits characteristics of a cancer/testis tumor like-antigen. Moreover, IL13-based bacterial toxin-containing recombinant anti-cancer cytotoxins are arguably the most potent anti-HGA agents; the first generation of these cytotoxins, hlL13-PE38QQR, has just entered Phase III clinical trials in the treatment of patients with recurrent GBM. IL13 structure-function relationship analyses revealed several micro-regions important for its interaction with the respective two types of receptors that are present in transformed cells (IL13Ralpha2) or normal tissues (IL13/4R). It was uncovered that alpha-helix D of the cytokine contains the region responsible for the binding of IL13 to IL13Ralpha2. Three amino acids within the alpha-helix D at positions 105, 106, and 109 were identified as a triad forming the binding region to IL13Ralpha2. Various substitution mutants of these residues produced IL13 moieties binding IL13Ralpha2 more avidly than the wild type cytokine. In this proposal, recombinant cytotoxins based on multiply mutated, specifically binding IL13Ralpha2 cytokines will be generated and examined. The optimal derivative of bacterial toxin, Pseudomonas exotoxin A, to be used in loco-regional treatment of HGA will be determined. Furthermore, the hypothesis that the levels of IL13Ralpha2 in HGA cells could be further increased and thus making them more susceptible to the killing by IL13 cytotoxins will be tested. To potentiate further their future clinical efficacy, combinatorial therapies involving IL13 cytotoxins will be developed. Preliminary data suggest that radiation increases the sensitivity of HGA cells to IL13 cytotoxin and that the IL13 cytotoxin sensitizes HGA cells to apoptosis-inducing agents. These results offer several novel opportunities to develop combinations of recombinant cytotoxins, radiation, and apoptotic agents in the treatment of HGA. Moreover, new ligands of double specificity targeting (cellular and intracellular) based on re-engineered IL13 will be generated. Peptidomimetics of IL13 binding to IL13Ralpha2 will also be a focus of these studies. Thus, this proposal aims at (i) optimizing IL13 cytotoxin efficacy either by remodeling the recombinant drug candidate or modulating expression of its target, or by creating combinatorial therapies, and (ii) more efficacious utilization of the IL13 system in malignancy by generating new types of ligands of multiple targeting specificity or small molecules of complete specificity for IL13Ralpha2.